Project Summary The goal of this project is to investigate how different interferon-stimulates genes (ISGs) protect different organs of mice from infection by specific RNA viruses. One focus will be on determining the in vivo antiviral properties of the three Ifit proteins, which are ISGs that are strongly induced in many tissues of virus-infected mice. A recent investigation by us has revealed that Ifit2, but not ifit1, knock-out mice are highly susceptible to neuropathy caused by infection of neurons of the peripheral and the central nervous system by a rhabdovirus, vesicular stomatitis virus (VSV). Here we propose to investigate the mechanism underlying the role of Ifit2 in protecting mice against VSV infection. In Aim 1, using two newly generated knock-out mouse lines, we will assess the abilities of Ifit3 or all Ifits in combination, to block pathogenesis caused by a number of RNA viruses, including VSV. Using the new Ifit2 conditional knock-out mice, we will identify the cell types in which Ifit2 expression is necessary for preventing neuropathogenesis caused by VSV infection. Using two approaches to achieve its cell-specific transgenic expression, we will also examine whether Ifit2 expression is sufficient for its preventive action or it requires help from other ISGs. In Aim 2, we will investigate the structure- function relationship of Ifit2; for this purpose we will extensively use the VSVTH mutant, which expresses Ifit2 from the Ifit2 gene built into the VSV genome itself. We will determine whether other mouse and human Ifits can substitute for Ifit2 in providing protection against VSV and which regions and properties of Ifit2 are required for its protective effect; moreover, we will identify the step of VSV-replication that is blocked by Ifit2. We observed that Ifit2 binds to RNAs containing AU-rich sequences. Because many cytokine mRNAs contain such sequences in their 3'UTRs, we will investigate the functional consequences of Ifit2 binding to them. Our investigations revealed that Ifit2 protects only the nervous system from VSV infection, whereas other ISGs protect other organs, such as the lung and the liver. Although in infected mice, Ifit2 is induced in non-neuronal cells as well, it is neither necessary nor sufficient for blocking VSV replication in them, indicating the existence of a neuron-specific partner of Ifit2. We will search for Ifit2's RNA or protein partners in infected neurons, using both in vitro and in vivo approaches. Our proposed investigation will illuminate the nature of the virus-, cell- and ISG-specific antiviral actions of IFN.